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Accelerated Analysis of On-Site

Pesticide Detection in Vegetables by


Agilent 5975T LTM GC/MSD and TSP
Application Note
Food Diagnostic

Authors

Abstract

Suli Zhao, Andy Zhai

Thermal separation probe (TSP) is a rapid, rugged, and inexpensive approach to gas

Agilent Technologies (Shanghai) Co., Ltd.

chromatography (GC) or gas chromatography mass spectrometry (GCMS) for analysis

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of semi-volatiles such as pesticides. With TSP, no sample cleanup is required to

Shanghai 200131

achieve quantitative and confirmatory results for quick, on-site detection of pesticides.

China

A fast method is established for the transportable 5975T LTM GC/MSD. With the help
of DRS software, we provide a good solution for accelerated analysis of pesticides for
on-site detection.

In this application note, an Agilent 5975T LTM GC/MSD was


evaluated to detect pesticides in vegetables that were pretreated, without a cleanup step, with the TSP tool and an isothermal
inlet. In complex extracts without the cleanup step, TSP
requires a selective detection technique to determine the analytes among the many semi-volatile matrix components.
Agilents DRS software and RTL function are good tools to
extract the targets from matrix semi-volatiles in a short time. A
5975T LTM GC/MSD with a quick ramp heating oven rate and
fast cooling cycle provides an ultra-fast sample cycle for this
application.

Introduction
Current methods used in the analysis of pesticide residues in
vegetables are time-consuming, labor intensive, costly, or do
not detect a wide range of analytes. Extraction of fruit and vegetables with acetone, acetonitrile or ethyl acetate is often followed by a clean-up step to remove co-extractives before gas
chromatographic analysis. In traditional method, clean-up is
necessary to prevent the build-up of nonvolatile matrix components in the injection liner and capillary column, and reduce the
rate of deterioration in chromatographic performance of the GC
system. For example, the U.S. Food and Drug Administration
(FDA) is responsible for the monitoring of vegetables in the
United States, and although they achieve a wide analytical
range, the methods require cleanup and solvent evaporation
steps prior to analysis using selective GC detectors. QuEChERS
has been demonstrated to quickly extract pesticide residues
from vegetables and save time in pretreatment process, but is
not very suitable for on-site analysis, which needs a quicker
cycle and much less solvent cost. Clean-up techniques such as
solid phase extraction (SPE), liquidliquid partitioning and gel
permeation chromatography (GPC) are often employed, but
increase the overall sample preparation time, increase the cost
of the method, and can result in the loss of pesticide recovery.

Highlights
TSP (Thermal separation probe)
Agilents RTL pesticides library and DRS software
Confirm the blind added multi-pesticides in 23 min after
sample running
Transportable 5975T LTM GC/MSD

Experimental
Software required
G1701EA GC/MS ChemStation (latest version)

TSP for GC injection is a technology that minimizes sample


preparation and still provides a rugged analytical approach for
complex matrices. TSP involves the placement of a small
amount of sample material or liquid extract into a 40 L disposable micro-vial. The sample and micro-vial are manually placed
into the GC/GCMS inlet and heated rapidly to thermally desorb
semi-volatile components, such as pesticides, in the sample. A
major benefit to the TSP approach is that nonvolatile matrix
components, which normally contaminate the GC liner and column in traditional injection approaches, remain in the microvial, which can be disposed after every injection. With the TSP
and its thermal extraction, only compounds that can be vaporized from the vial are introduced into the column. There were
many applications on similar probes in the past with a programmed temperature injector.[1]

G1716 MSD Deconvolution Reporting Software


(Version A.04.00 or newer)
G1033A NIST08 Mass Spec Library + AMDIS + NIST
Library Search
G1675AA Japan Positive List

Reagents and chemicals


All reagents were analytical or HPLC grade. The pesticides
were purchased from Sigma-Aldrich (St. Louis, MO, USA).
The water was from a MilliQ system (Milford, Mass, USA).

Equipment and Materials


This experiment was performed on an
Agilent 5975T LTM GC/MSD. Extraction was achieved with
Agilent SampliQ QuEChERS AOAC Extraction kits
(p/n 5982-5755, Agilent Technologies Inc.,
Wilmington, DE, USA).

1% HAc in ACN was added to each tube using the dispenser.


An Agilent SampliQ QuEChERS extraction salt packet from the
kit (p/n 5982-5755), containing 6 g of anhydrous MgSO4, and
1.5 g of anhydrous NaOAc, was added directly to the tubes.
The salt bag was massaged carefully to break up any salt
clumps before pouring. The tubes were examined to ensure
that no powder was left in the threads or rims of the tubes.
Sample tubes were sealed tightly and shaken vigorously for 1
min by hand to ensure that the solvent interacted with the
entire sample and crystalline agglomerates were dispersed.
Sample tubes were centrifuged at 4,000 rpm for 5 min. The
liquid layer was taken for GCMS injection.

Instrument conditions
Table1.

Instrumentation and Conditions of Analysis

Instrumentation
GCMS system
Inlet
Column
Guard column

Agilent 5975T LTM GC/MSD


Split/splitless (liner: 5062-3587)
HP-5 ms LTM 10 m 0.18 mm 0.18 m
1 m column with same phase as analytical
column, connected to the injector.

Experimental conditions
Inlet temperature
Injection volume
Injection mode
Carrier gas
Head pressure
LTM oven temperature

260 C
1L
splitless; purge after 1min: 100 mL/min
helium
1.6 mL/min, constant flow mode
50 C (0.2 min), 125 C /min, 125 C (0 min),
50 C/min, 300 C (2 min)
Method
RTlocked to chlorpyrifos methyl at 2.70 min
Transfer line temperature 260 C
MSD interface
Ion source
Quad. temperature
Ionization mode
Scan mode
EMV mode
Gain factor
Resulting EM voltage
Solvent delay

Results and discussion


There is increasing pressure to reduce costs in pesticide testing and increase productivity without sacrificing analytical
quality. TSP has been tested as a tool for reducing sample pretreatment time. We used extractions of tomato, cucumber, and
pepper to test the TSPs pesticide detection capabilities, including peak discrimination, repeatability, and so on. The LTM column rapidly heats up and cools down column temperature, further reducing run time and cycle time.

270 C
230 C
150 C
EI
full scan, 50550 m/z
Gain factor
5.00
1129 V
0.5 min

Fast methods established for on-site pesticide


detection with TSP
An Agilent GC MXLATOR software tool was used to find operating conditions for the faster methods. The faster methods
were scaled exactly as predicted, using a combination of
Agilents method translation (MTL) and RTL software. Because
scaling was exact, these faster methods could be used with
precisely scaled pesticide libraries, making the screening
process even more powerful and adaptable to individual needs
[2]. With this software, we obtained the fast method from a
method with a 30 m, 0.25 mm, 0.25 m column to a method
with a 10 m, 0.18 mm, 0.18 m column, to obtain a fivefold
shortened running time. The original method was based on tha
Japanese positive list method [3]. We also shortened the retention index of relative compounds in the library with a fivefold
time reduction. The application of all the results in this paper
was retention times locked to corresponding requirements.

Sample preparation
Samples
Organically grown, pesticide-free cucumbers, tomatoes, and
green peppers were purchased from a local grocery store. The
samples were spiked to different concentration levels with a
certain number of pesticides.

Sample preparation
Extraction/Partitioning
Approximately one pound of cucumbers or tomatoes were
chopped into small, bean-sized cubes. Two ceramic homogenizers (p/n 5982-9313) were placed into a 50 mL centrifuge tube
(from the SampliQ QuEChERS extraction kit) and a 15 g ( 0.1g)
amount of previously homogenized sample was placed into the
same tube. QC samples were added with 100 L of appropriate
QC spiking solution. A 100 L amount of internal standard spiking solution was added to all samples except the control blank.
Tubes were capped and vortexed for 1 min. A 15 mL amount of

With the fast method, we tested the TSP availability for the
pesticides including organo-phosphorous, organo-chlorine and
pyrethroid pesticides. The added concentration was 5.0 g/L
and injected volume was 1 L. Figure 1 shows that TSP injection can produce good peak shapes for all different types of
pesticides with no peak discrimination.

1
2
3
4
5
6
7
8
9
10
11
12

Figure1.

Dichlorvos
Omethoate
Hexachlorobenzene
Chlorpyrifos methyl
Quinalphos
p,p-DDE
Ethion
Phosalone
Fenvalerate I
Fenvalerate II
Deltamethrin I
Deltamethrin II

TIC of 12 pesticides in cucumber extraction.

extraction were some sugars, vitamins, and pigments. Most of


them remained in the vial and only volatile compounds that can
be vaporized from the vial were introduced into the system. At
20 injections, the vial became dark, but the liner remained
clean; this shows that the TSP has the capability to trap the
heavy matrix, which would be removed with a clean-up step. In
this test, the running time was extended for higher boiling compounds. We tested one baseline every 10 injections. Figure 2
compares five baselines. It shows that the system could stay
clean after the rich interference sample was injected. The sample was extracted without a clean-up step, showing TSP could
be used for the no-clean-up sample test and save more sample
preparation time.

Repeatability test with TSP


In order to evaluate the stability of the system we tested
17 organo-chloro pesticide solutions in acetone at a 1.0 g/mL
concentration. We continuously injected eight times and the
RSD% of all the compounds was 2.3 ~7.8% with 1 L injection
volume. The RSD results may have been affected by the manual syringe volume. The results could satisfy the requirements of
on-site analysis.

Keeping clean capability test with TSP


We tested the baseline of 50 continuous injections of tomato
extractions without a clean-up step. The main compositions of

TIC: Baseline D\data.ms

TIC: Baseline 2.D\data.ms

TIC: Baseline 3.D\data.ms

TIC: Baseline 4.D\data.ms

TIC: Baseline 5.D\data.ms

Figure 2.

Baselines of the 50 injections test.

Blind pesticide spiked samples and Real sample


test with TSP

nine organo-phosphorous pesticides (100 ng/mL each). Table 2


is the DRS report of the results.

Three samples of vegetable extracts were spiked with different


pesticides at different concentration levels. Eighty spiked pesticides were tested in three samples. With the TSP injection and
AGILENT DRS software, most of the tested pesticides could
be confirmed when the concentration level was higher than
100 ppb except acephate and methamidophos. When the concentration was increased to 500 ppb, all of the tested pesticides could be confirmed by DRS. Figure 3 shows a chromatogram of a mixture of three samples that was spiked with

Three pesticides were identified and confirmed by DRS in a


tomato extract that was not spiked. They were Pyrimethanil,
Procymidone and Dimethomorph. Figure 4 shows a spectra
report of procymidone from AMDIS software. It shows that it
would not identify the compounds by using only a library
search, without deconvolution. All the results show that with
the fast method and TSP, we can identify unknowns with the
help of DRS library.

Figure 3.

TIC of 100 ppb pesticides spiked mixture of three samples.

Table 2.

DRS for the Mixture of Three Samples Whose Chromatogram is Shown in Figure 3
Amount (PPB)

R.T.

Cas number

Compound name

1.3748

62737

Dichlorvos

2.2261

13194484

2.3503

Chemstation

AMDIS

AMDIS

NIST

Match

R.T. Diff sec.

Reverse match

Hit num.

98

79

0.1

74

Ethoprophos

98

96

0.2

89

298022

Phorate

100

97

0.6

92

2.5501

333415

Diazinon

100

78

0.2

74

2.7639

298000

Methyl parathion

100

92

0.4

81

2.8992

121755

Malathion

100

77

0.1

81

2.9245

2921882

Chlorpyrifos

98

93

0.3

87

2.9565

56382

Parathion

100

79

0.3

77

3.1993

961115

Tetrachlorvinphos

98

83

0.1

82

Figure 4.

Deconvolution spectrum of procymidone in unspiked tomato extraction.

2. C. Kai Meng; Fast Screening of Pesticides and Endocrine


Disrupters Using the Agilent 6890/5973N GC/MSD
System, Part II, Agilent Technologies publication
5980-1057EN.

Conclusion
An Agilent 5975T LTM GC/MSD with TSP solution produced
good results for on-site detections, meeting the requirements
for on-site applications, such as quick response and fast identification. TSP can save sample pretreatment time and keep the
system clean. Agilent DRS software helped extract the target
compounds spectrum from the matrix interface and create a
fast running method. This three-way combination provides a
good solution for accelerated analysis of pesticides, especially
for on-site detection.

3. Philip L. Wylie; Screening for Pesticides in Food Using the


Japanese Positive List Pesticide Method: Benefits of
Using GCMS with Deconvolution Reporting Software and
a Retention Time Locked Mass Spectral Database, Agilent
Technologies publication 5989-7436EN

For More Information

Reference

For more information on our products and services, visit our


Web site at www.agilent.com/chem.

1. Hongwu Jing and Aviv Amirav*; Pesticide Analysis with


the Pulsed-Flame Photometer Detector and a Direct
Sample,
*Introduction Device; Anal. Chem. 1997, 69, 1426-1435
7

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Information, descriptions, and specifications in this
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Agilent Technologies, Inc., 2011


Printed in the USA
June 8, 2011
5990-8067EN